PROJECT SUMMARY/ABSTRACT To date, adoptive T cell therapy is a promising approach to reactivate the patient's own immune system to fight against tumors. While adoptive transfer of tumor-infiltrating lymphocytes (TILs) enriched with cytotoxic CD8+ T cells (CTLs) results in a 50% response rate in multiple phase II clinical trials in metastatic melanoma, CD8+ T cell transfer alone does not improve clinical response suggesting a role of helper CD4+ T cells. There is increasing evidence on the potential function of a CD4+ T helper subset, named as T follicular helper (Tfh) cells in triggering anti-tumor activity; however, the role of Tfh cells in tumor immunity has not been elucidated. Since Tfh-derived cytokine interleukin (IL)-21 is essential for activity and maintenance of CTLs, understanding their function in tumor microenvironment could be invaluable for further improvement of current immunotherapy approaches. Our preliminary results reveal the potential role of Tfh cells in the control and prevention of melanoma tumor growth. Both pre-clinical murine melanoma tumor studies and patient studies show correlation between the presence of intratumoral Tfh cells with reduced tumor growth and improved survival, suggesting that Tfh cells are responsible for the promotion of anti-tumor immunity. Interestingly, adoptive transfer of antigen specific Tfh cells into melanoma bearing wild-type mice results in increased number of intratumoral CTLs, as well as in efficient tumor eradication, suggesting the role of Tfh cells in promoting CTL expansion and anti-tumor reactivity. Therefore, we propose to investigate the therapeutic potential of Tfh cells for melanoma treatment and identify regulatory mechanisms whereby Tfh cells contribute to anti-tumor immunity. In Aim 1, we will determine the mechanism(s) whereby tumor antigen-specific Tfh cells contribute to anti-tumor immunity by utilizing Tfh cells from Bcl6-reporter mice crossed with tyrosinase-related protein 1 (TRP-1) (melanoma antigen) T-cell receptor (TCR) transgenic mice. In addition, we will undercover the mechanism(s) responsible for maintenance of Tfh cells in the tumor site. In Aim 2, we will develop an optimal method for culturing and expansion of Tfh TIL lines from melanoma patients and determine their function towards autologous CD8+ TILs and tumor cells in vitro and in vivo in an NSG mouse xenograft model of melanoma. Overall, the proposed research will help to understand the therapeutic potential of Tfh cells for melanoma treatment and identify regulatory mechanisms whereby Tfh cells contribute to anti-tumor immunity. The implications from this work are significant since the results will provide the basis for potential usage of these cells for improvement of current immunotherapy approaches against melanoma.